Down-regulation of STAT1α target genes in STAT1β overexpressing cells. (A) Inhibition of the transcriptional activity of STAT1α by overexpression of STAT1β. Cells were cotransfected with a plasmid construct containing the luciferase gene driven by the TAP1 promoter with an inactivated κB site. Cells were transfected with an empty CKR516 vector (empty), with the CKR516-STAT1β vector, or with the CKRSTAT1α vector. The experiment was repeated 3 times with identical results. (B) Purification of EGFP-inducible and noninducible cells transfected with the CKR516-STAT1β vector. Twenty-four hours of treatment of cells with 0.6 μg/mL doxycycline yielded a heterogeneous cell population (top graph) containing EGFP-positive (top gate) and -negative cells (bottom gate). A typical result of EGFP-positive and -negative cell sorting by flow cytometry is shown in the bottom graphs. The percentage of cells in the 2 gates is indicated in each graph. (C) RQ-PCR analysis of STAT1 target genes in CKR516-STAT1β–transfected cells. Levels of p21WAF1/CIP1, IRF1, PKR, and TAP1 mRNA were measured by RQ-PCR in extracts from EGFP-positive and -negative sorted cells and compared to the level of Abl1 mRNA. (D) Detection of the c-myc protein in CKR516-STAT1β–transfected cells. Detection of c-myc was performed by Western blot analysis of total protein extracts from cells treated (+) or not treated (–) with 0.6 μg/mL doxycycline for 24 hours.